Methionine metabolism occupies a central role in cellular chemistry. The metabolic and regulatory importance of its chief product, S-adenosylmethionine (AdoMet), has long been known, and includes such biologically important functions as methylation, polyamine biosynthesis, side-chain donation, and allosteric enzyme regulation (Castoni et al., The Biochemistry of Adenosylmethionine, Columbia University Press, New York, pp.557-577 (1977)). The most frequent metabolic fate of AdoMet is transmethylation. This reaction forms homocysteine, which may be either recycled to methionine by remethylation (Finkelstein et al., J. Biol. Chem., 259, 9508-9513 (1984)) or used to synthesize cysteine by transsulfuration (Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, pp. 693-734 (1989)). Each of these pathways consumes about half of the intracellular homocysteine, thus they account for the metabolism of all of this metabolic byproduct Finkelstein et al., J. Biol. Chem., 259, 9508-9513 (1984; and, Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, pp. 693-734 (1989)).
Cystathionine .beta.-synthase (EC 4.2.1.22) (CBS) catalyzes the first irreversible step of homocysteine transsulfuration. This enzyme conjugates homocysteine and serine forming cystathionine, which is subsequently converted into cysteine and .alpha.-ketobutyrate in the cystathionine .gamma.-lyase reaction (Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGrawHill, New York, pp. 693-734 (1989)). Pyridoxal 5'-phosphate is a cofactor for these reactions (Kraus et al., J. Biol. Chem., 253, 6523-6528 (1978) and AdoMet enhances the affinity of the enzyme for homocysteine by allosteric activation (Roper et al., Arch. Biochem. Biophys., 298, 514-521 (1992)). Posttranslational proteolysis similarly affects the affinity of the synthase for homocysteine (Skovby et al., J. Biol. Chem., enzyme responds to joint administration of glucocorticoids and cyclic AMP enhancers (Goss, J. Cell. Sci., 82, 309-320 (1986)). These regulatory parameters are consistent with its role as a committed step in a branch-point of metabolism.
Deficiency of synthase in humans is the leading cause of homocystinuria (Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, pp. 693-734 (1989)). Untreated patients develop a number of phenotypic traits which include skeletal abnormalities, dislocated optic lenses, mild to profound mental retardation, and vascular disorders (Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, pp. 693-734 (1989)). Some patients respond to vitamin B.sub.6 administration while others are unresponsive to this therapeutic intervention (Mudd et al., The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, pp. 693-734 (1989); and, Lipson et al., J. Clin. Invest., 66, 188-193 (1980)). A growing body of evidence now suggests that vascular disorders found in one-third of the patients with premature arterial disease or cerebrovascular disease are the result of mild hyperhomocysteinemia some of which may be due to heterozygous CBS deficiency (Boers et al., N. Engl. J. Med., 313, 709715 (1985); and, Clarke et al., N. Sngl. J. Med., 324, 1149-1155 (1991)). In addition, the CBS gene maps to human chromosome 21 at q 22.3 (Munke et al., Am. J. Hum. Genet., 42, 550-559 (1988)). This region of chromosome 21 is evidently associated with many Down syndrome features; microduplications in this region precipitate many of the features associated with Down phenotype (Korenberg et al., Am. J. Hum. Genet., 43, A110 (1988); and, Korenberg et al., A. J. Hum. Genet., 47, 236-246 (1990)). Since the coding region of human CBS has not heretofore been determined, it is necessary to determine its sequence prior to detecting mutations in patients with CBS deficiency. Also, only through recombinant techniques can large quantities of CBS be made available as replacement enzyme to treat patients suffering from homocystinuria and other diseases resulting from CBS deficiency.
Therefore, a need arose to purify and isolate DNA sequences of CBS for evaluation of mutations in patients and for obtaining CBS in large quantities. One way to isolate a DNA sequence encoding CBS is via cDNA cloning. In this process, messenger RNA (mRNA) is isolated from cells known or suspected of producing the desired protein. Through a series of enzymatic reactions, the mRNA population of the cells is copied into a complementary DNA (cDNA). The resulting cDNA is then inserted into cloning vehicles and subsequently used to transform a suitable prokaryotic or eukaryotic host. The resultant gene library is comprised of a population of transformed host cells, each of which contain a single cDNA or cDNA fragment. The entire library, therefore, provides a representative sample of the coding information present in the mRNA mixture used as a starting material.
cDNA libraries are screened using specific nucleic acid or antibody probes. Nucleic acid probes are useful for locating cDNAs by hybridization and autoradiography techniques. This approach, however, requires previous knowledge of at least a portion of the protein's amino acid or DNA-encoding sequence. Alternatively, methods have been developed to identify specific clones by probing recombinant cDNA libraries with antibodies specific for the encoded protein of interest. This method can be used with "expression vector" cloning vehicles since elaboration of the product protein is required. An example of this is the bacteriophage .lambda.-gt11 system described by Young and Davis, Proc. Natl. Acad. Sci., 80, 1194-1198 (1983).
Once the cDNA is purified and isolated, the full length cDNA sequence can be used for insertion into expression vectors. This leads to the production of active enzyme.